Cystic Fibrosis (CF) is the most common lethal autosomal recessive disease in Caucasians and is caused by defects in the cystic fibrosis conductance regulator (CFTR) chloride channel. Although multiple organs are affected in CF, persistent bacterial infections in the lung are the most life-threatening component of the disease. The development of treatments for CF lung disease has been hindered by the lack of CF animal models capable of reproducing the natural progression of lung disease in CF patients. We recently generated a CFTR-knockout ferret model that reproduces human disease phenotypes in the lung, pancreas, liver, intestine, and vas deferens. The CFTR-knockout ferrets demonstrate two lung colonization phenotypes that will be useful in developing therapies for CF-rapidly lethal bacterial infections of the lung during the during the early neonatal period and a slower progressive bacterial colonization of the lung that leads to death by 8 months of age. We seek to use this new model to develop recombinant adeno-associated virus (rAAV) gene therapies for CF lung disease. Important biologic problems to be addressed include: 1) characterization of a novel inhibitory protein found in human and ferret airway secretions, which tightly binds to the rAAV1 capsid and inhibits in vivo gene transfer, and dissection of its intracellular proteasome-dependent mechanisms of action, 2) the generation of rAAV-CFTR vectors that are capable of reversing lung disease in the CF ferret model and compatible with both packaging limitations of the rAAV genome and cellular requirements for efficient CFTR functional complementation, and 3) identification of the sites in the lung (surface airway epithelium vs submucosal glands) that must be targeted for effective complementation of CF lung disease. The third goal of the proposal draws on the unique ability of the Engelhardt laboratory to rapidly generate cloned CFTR-knockout ferrets that transgenically express recombinant fCFTR at biologically relevant cellular sites the lung. Thus, this proposal addresses novel viral and cellular mechanisms that are relevant to improving gene therapies to the airway, while also tackling difficult cell biology questions about the pathogenesis and treatment of CF lung disease. This proposal will significantly enhance the field's ability to effectively develop therapeutic strategies for treatment of the CF lung, not only using rAAV vectors, but also other pharmacologic and gene-based therapies. PUBLIC HEALTH RELEVANCE: Cystic Fibrosis (CF) is the most common life-threatening autosomal recessive condition among Caucasians, with over $450 million dollars spent annually on clinical care of CF patients in the U.S. alone. Despite the fact that the gene defect responsible for CF was discovered over 20 years ago, a cure for CF has yet to emerge- in large part due to a lack of appropriate animal models for use in studying the disease process and testing therapies. Using new CF animal models, this proposal will determine the cellular sites in the CF lung that must be corrected to prevent lethal lung infections, as well as develop viral vectors capable of delivering therapeutic expression of the CFTR gene to the CF lung.